Mobile genetic elements have profoundly influenced the evolution of bacterial pathogens. Many determinants of virulence and antibiotic resistance are borne by mobile elements, such as plasmids and bacteriophages, which are capable of horizontal transmission. During the current grant cycle, we explored several fundamental questions regarding the molecular biology of two mobile genetic elements, CTXqj and SXT, which have profoundly influenced the pathogenicity and evolution ofthe cholera pathogen. Vibrio cholerae. Addressing these aims has yielded observations that suggest previously unrecognized mechanisms of bacteriophage gene regulation and new insights regarding the biology and evolution of Integrative Conjugative Elements (ICEs). Furthermore, knowledge gained from these studies proved to be critical for our studies of the origin of the V. cholerae strain that gave rise to the cholera outbreak in Haiti. During this grant cycle, we also expanded the scope of our work to include investigation of new aspects of V. cholerae biology and pathogenicity. In particular, we developed a new infant rabbit model of disease that closely resembles human cholera. We will exploit this model and new high throughput tools we have developed to address three new aims during the MERIT extension period: 1) Assess the spatial and temporal patterns of V. cholerae gene expression during infection; 2) Identify and characterize the determinants of V. cholerae transmission; and 3) Characterize the innate immune response to V. cholerae intestinal colonization. Completion of these studies will provide the most comprehensive knowledge ofthe course of gene expression during infection for any bacterial pathogen. Additionally, it will enhance our understanding of the processes underlying V. cholerae pathogenicity and transmission. Finally, these studies should provide insight into the innate immune response within the intestinal tract, modulation of which has profound ramifications both for normal intestinal homeostasis and for disease. Collectively, these studies will yield valuable new knowledge for the creation of new antimicrobial agents and vaccines.